The broad objective of this proposal is to understand both the biochemical mechanism and biological function of DNA helicases, i.e., how they translocate along single-stranded DNA (ssDNA) to unwind doublestranded DNA (dsDNA) and, how this effort is translated into biological function (i.e., useful work). DNA helicases are ubiquitous and essential for cellular function. These enzymes "unwind" dsDNA into its component DNA strands, in a reaction requiring nucleoside triphosphate hydrolysis. DNA helicases are motor proteins that travel along DNA as part of their unwinding function. The rates, distances, and consequences of nucleic acid unwinding differ, but are all related to the biological function of these proteins; hence their mechanisms of action vary. This grant proposal has two broad specific aims. The first is to study a "complex" catalytic helicase, the RecBCD enzyme. RecBCD enzyme is a multi-subunit, multifunctional enzyme that possesses both helicase and nuclease activities. It can recognize, while translocating, a specific DNA sequence called khi. The khi sequence regulates the nucleolytic and translocation activities of the RecBCD enzyme, and it directs the loading of the RecA protein onto the ssDNA produced. Hence, RecBCD enzyme is a unique helicase that responds to a signal sequence embedded within dsDNA. The second aim is to study a seeming "simple" helicase, RecQ. The RecQ helicase interacts specifically with topoisomerase III to effect unexpected changes in DNA topology. Understanding how these motor proteins accomplish these tasks is the overall goal of this proposal. To accomplish this goal, biochemical, enzymatic, structural-functional, and single-molecule analyses are planned. This research will provide basic information about protein-DNA interactions; insight into the behavior of molecular motors, their translocation process, and their application as "nanomachines"; and an appreciation of the molecular events responsible for aberrant cellular processes. Mutations in genes that encode putative helicases are associated with human syndromes such as xeroderma pigrnentosum (ERCC2 and ERCC3); Cockayne's (ERCC6); Bloom's (BLM); Werner's (WRN;) and Rothmund-Thomson (RecQ4), showing that an understanding of these proteins is crucial for the understanding of disease processes as diverse as cancer and premature aging.